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  • br ROS originated in mitochondria rather than from

    2019-10-21


    ROS originated in mitochondria, rather than from other in-tracellular sources, are confirmed as one of the most pathogenic factors when the rate of their production exceeds the homeostatic level [145,146]. And enhanced mitochondrial ROS production leads to  Journal of Inorganic Biochemistry 190 (2019) 45–66
    mitochondrial permeability transition pore (mPTP) opening with con-sequential dissipation of mitochondrial transmembrane potential (MTP) and initiation of cell death [140]. Collapse of MTP represents instant change in permeability of the inner mitochondrial membrane that permits to molecules of a size up to 1.5 kDa to go through the mem-brane. The following course of events includes interruption of ATP synthesis, mitochondrial swelling, and initiation of cell death. In order to evaluate if enhanced generation of O2%− due to treatments with 1 and CDDP induces mPTP opening, we assessed the MTP status in treated cells.
    With reference to O2%− production in CDDP-treated AsPC-1 samples (Fig. 6), it was expected the same treatment would not cause significant impact on MTP (Fig. 7). However, it could not be predicted that CDDP acted as MTP stabilizing agent, decreasing incidence of spontaneously developed alteration of MTP seen in non-treated AsPC-1 cells. Yet the same phenomenon was found in MCF-7 Aztreonam subjected to CDDP (Fig. 8). While half of MCF-7 cells treated with CDDP were positive for increased O2%− with almost twice-greater MFI, percentage of cells with dissipated MTP was also below those of non-treated samples like for AsPC-1. Re-cently, Marullo et al. [120] showed that mitochondrial ROS generation is another important mechanism responsible for anticancer activity of CDDP yet not well characterized previously. In their work, significant increase in mitochondrial O2%− levels starts after 16 h of CDDP treat-ment indicating that time course of O2%− production is consistent with a reduced synthesis of electron transport chain proteins due to forma-tion of adducts with mitochondrial DNA [147]. Therefore, according to previously postulated mechanism, increased extent in mitochondrial O2%− is rather delayed, as a consequence of disrupted protein synthesis, instead of an early event caused by pro-oxidant activity of CDDP. This is not in agreement with our observation in MCF-7 cells (Fig. 6). Since in the current investigation CDDP is employed only as a reference com-pound, further discussion on possible mechanisms underlying elevated mitochondrial O2%− formation while preserved MTP (Figs. 6–8), would be beyond the scope of our study. Final remark concerns the obvious difference in mechanisms of activity between CDDP and investigated cadmium complex at the mitochondrial level.
    3.4.5. Complex 1 shows a mighty activity on 3D CSC models Significance and advantages of drug activity testing on 3D models
    compared to 2D cultures have been reviewed previously [117,147]. Here, we tested activity of 1 on AsPC-1 and MCF-7 3D cultures over 8 days treatment time, with CDDP as a reference drug. Gained results provide an excellent illustration of inconsistency between drug's effects obtained on 2D and 3D models, additionally emphasizing the im-portance that use of in vitro tumors has for more accurate validation of preclinical drug development [148,149]. While 1 in 2D MCF-7 model displayed vigorous pro-apoptotic activity already at 10 μM (Fig. 3), it achieved growth inhibition of MCF-7 spheroids only at the highest applied concentration of 100 μM (Figs. 9 and 10). While treatment with 1 at 1 μM did not show any effects, at 10 μM a modest activity with a 2.1 ± 0.1-fold in size increase as compared to 2.5 ± 0.3-fold in non-treated control on day 8.
    For CDDP, examined on MCF-7 3D model previously [150], the current study confirms its ability to reduce initial size of treated spheroids at concentrations of 10 μM (0.85 ± 0.04-fold) and 100 μM